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Emerging Microbes & Infections

ISSN: (Print) 2222-1751 (Online) Journal homepage: https://www.tandfonline.com/loi/temi20

Middle East respiratory syndrome coronavirusinfection in non-camelid domestic mammals

Ahmed Kandeil, Mokhtar Gomaa, Mahmoud Shehata, Ahmed El-Taweel,Ahmed E. Kayed, Awatef Abiadh, Jamel Jrijer, Yassmin Moatasim, OmniaKutkat, Ola Bagato, Sara Mahmoud, Ahmed Mostafa, Rabeh El-Shesheny,Ranawaka APM Perera, Ronald LW Ko, Nagla Hassan, Basma Elsokary, LotfiAllal, Ahmed Saad, Heba Sobhy, Pamela P. McKenzie, Richard J. Webby, MalikPeiris, Mohamed A. Ali & Ghazi Kayali

To cite this article: Ahmed Kandeil, Mokhtar Gomaa, Mahmoud Shehata, Ahmed El-Taweel,Ahmed E. Kayed, Awatef Abiadh, Jamel Jrijer, Yassmin Moatasim, Omnia Kutkat, Ola Bagato, SaraMahmoud, Ahmed Mostafa, Rabeh El-Shesheny, Ranawaka APM Perera, Ronald LW Ko, NaglaHassan, Basma Elsokary, Lotfi Allal, Ahmed Saad, Heba Sobhy, Pamela P. McKenzie, RichardJ. Webby, Malik Peiris, Mohamed A. Ali & Ghazi Kayali (2019) Middle East respiratory syndromecoronavirus infection in non-camelid domestic mammals, Emerging Microbes & Infections, 8:1,103-108, DOI: 10.1080/22221751.2018.1560235

To link to this article: https://doi.org/10.1080/22221751.2018.1560235

© 2019 The Author(s). Published by InformaUK Limited, trading as Taylor & FrancisGroup, on behalf of Shanghai ShangyixunCultural Communication Co., Ltd

Published online: 16 Jan 2019.

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Middle East respiratory syndrome coronavirus infection in non-cameliddomestic mammalsAhmed Kandeila, Mokhtar Gomaaa, Mahmoud Shehataa, Ahmed El-Taweela, Ahmed E. Kayeda,Awatef Abiadhb, Jamel Jrijerb, Yassmin Moatasima, Omnia Kutkata, Ola Bagatoa, Sara Mahmouda,Ahmed Mostafaa,c, Rabeh El-Sheshenya,d, Ranawaka APM Pererae, Ronald LW Koe, Nagla Hassanf,Basma Elsokaryf, Lotfi Allalg, Ahmed Saadg, Heba Sobhyg, Pamela P. McKenzied, Richard J. Webbyd,Malik Peirise, Mohamed A. Alia and Ghazi Kayalih,i

aCenter of Scientific Excellence for Influenza Virus, National Research Centre, Giza, Egypt; bNature Link, Sfax, Tunisia; cInstitute of MedicalVirology, Justus Liebig University Giessen, Giessen, Germany; dSt. Jude Children’s Research Hospital, Memphis, TN, USA; eSchool of PublicHealth, University of Hong Kong, Sandy Bay, Hong Kong; fGeneral Organizations of Veterinary Services, Ministry of Agriculture and LandReclamation, Giza, Egypt; gFood and Agriculture Organization of the United Nations, Emergency Center for Transboundary Animal Diseases,Giza, Egypt; hHuman Link, Baabda, Lebanon; iUniversity of Texas Health Sciences Center, Houston, TX, USA

ABSTRACTDromedary camels are natural host of the Middle East respiratory syndrome coronavirus (MERS-CoV). However, there arelimited studies of MERS-CoV infection of other domestic mammals exposed to infected dromedaries. We expanded oursurveillance among camels in Egypt, Tunisia, and Senegal to include other domestic mammalian species in contact withinfected camels. A total of 820 sera and 823 nasal swabs from cattle, sheep, goats, donkeys, buffaloes, mules, and horseswere collected. Swabs were tested using RT-PCR and virus RNA-positive samples were genetically sequenced andphylogenetically analysed. Sera were screened using virus microneutralization tests and positive sera (where available)were confirmed using plaque reduction neutralization tests (PRNT). We detected 90% PRNT confirmed MERS-CoVantibody in 35 (55.6%) of 63 sera from sheep collected from Senegal, two sheep (1.8%) of 114 in Tunisia and a goat(0.9%) of 107 in Egypt, with titres ranging from 1:80 to ≥1:320. We detected MERS-CoV RNA in swabs from threesheep (1.2%) of 254 and five goats (4.1%) of 121 from Egypt and Senegal, as well as one cow (1.9%) of 53 and threedonkeys (7.1%) of 42 from Egypt. Partial sequences of the RT-PCR amplicons confirmed specificity of the results. Thisstudy showed that domestic livestock in contact with MERS-CoV infected camels may be at risk of infection. Werecommend expanding current MERS-CoV surveillance in animals to include other livestock in close contact withdromedary camels. The segregation of camels from other livestock in farms and live animal markets may need to beconsidered.

ARTICLE HISTORY Received 17 October 2018; Revised 26 November 2018; Accepted 3 December 2018

KEYWORDS MERS-CoV; surveillance; serology; Egypt; Tunisia; Senegal; sheep

Introduction

The Middle East Respiratory Syndrome (MERS) iscaused by a beta-coronavirus (CoV) first detected ina Saudi male in 2012 [1]. Since then, the World HealthOrganization (WHO) has recorded 2260 laboratory-confirmed cases, at least 803 of these being fatal [2].Surveys of camels in the Middle East and Africashowed that they are a natural host of MERS-CoV[2,3] and they are a source of human infection [4].

Host specificity of MERS-CoV is determined by thepresence of the dipeptidyl peptidase-4 (DPP4) recep-tors expressed on cell surfaces. DPP4 was found to bea functional receptor for the receptor-binding S1domain (RBD) of the MERS-CoV spike protein [5,6].There is limited data on the susceptibility of other

livestock in close contact with camels to MERS-CoVinfection. A survey of goats, camels, and sheep fromJordan showed no evidence of infection [7]. Surveyson horses in the United Arab Emirates, Saudi Arabia,and Oman did not provide conclusive evidence ofinfection [8,9].

We expanded our MERS-CoV surveillance pro-gramme in Egypt, Tunisia, and Senegal to includeother domestic livestock in contact with camels.

Results

We carried out 17 sampling visits to mixed farms orherds where camels and other livestock were raised inSenegal and Egypt and a livestock market in Tunisia.

© 2019 The Author(s). Published by Informa UK Limited, trading as Taylor & Francis Group, on behalf of Shanghai Shangyixun Cultural Communication Co., LtdThis is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricteduse, distribution, and reproduction in any medium, provided the original work is properly cited.

CONTACT Mohamed A. Ali mohamedahmedali2004@yahoo.com Center of Scientific Excellence for Influenza Virus, National Research Centre,El Buhouth Street, Dokki, Giza 12311, Egypt; Ghazi Kayali ghazi@human-link.org Human Link, Camelia II Building, Said Freiha Street, Hazmieh, 1003Baabda, Lebanon

Supplemental data for this article can be accessed at https://doi.org/10.1080/22221751.2018.1560235.

Emerging Microbes & Infections2019, VOL. 8https://doi.org/10.1080/22221751.2018.1560235

Camels in contact with these livestock were sampledduring these visits and MERS-CoV RNA was detectedin camels in 13 of the 17 sampling trips while seropo-sitive camels were detected in all trips (data notshown).

In total, 820 serum samples of livestock other thancamels in contact with camels were tested for MERS-CoV antibodies by the screening microneutralization(VMN) test and 56 of them had detectable antibodyto MERS-CoV. All but 2 cattle sera were from adultanimals. Out of these sera, 49 were available for confi-rmatory testing by PRNT tests using the stringent 90%reduction of plaque numbers (PRNT90) as the cri-terion for evidence of neutralizing antibody. Therewas PRNT90 antibody to MERS-CoV in 38 adult ani-mal sera; in 35 (55.6%) of 63 sheep sampled in Senegal,one of 107 goats sampled in Egypt and two of 114sheep sampled in Tunisia (Table 1). The PRNT90 anti-body titres of these positive sera ranged from 1:80 to≥1:360. Sera positive for MERS-CoV antibodies (37sheep sera from Senegal, one sheep from Tunisia andone goat from Egypt) with antibody titres rangingfrom 1:20 to >1:320 were selected for testing by micro-neutralization tests for bovine coronavirus (BCoV).Ten of the MERS-CoV antibody positive sheep serafrom Senegal and the sheep serum from Tunisia hadno detectable antibody to bovine coronavirus. Eventhose sera with antibody to bovine coronavirus hadantibody titre that were lower than that observed forMERS-CoV (Table S1).

MERS-CoV RNA was detected in 12 of 823 swabscollected from sheep, goats, cattle, buffalo, horses, don-keys and mules (Table 1), virus RNA being detected inthree sheep, five goats, three donkeys and one cowfrom Egypt or Senegal. All animals were adults exceptfor 1 juvenile goat and another juvenile donkey. Allswabs that tested positive by upE rtRTPCR had CTvalues ≥38 indicating low virus load and wereconfirmed by at least one additional RT-PCR assay

targeting the ORF1a, N or S genes (Table 2). Four ofthese upE rtRT-PCR positive samples were ORF1artRTPCR positive while the others were confirmed byN or S (RBD) gene RT-PCR assays. Five sampleswere also confirmed by sequencing the RBD regionof the spike gene. The host species of each MERS-CoV positive sample were confirmed by DNA barcod-ing to exclude possibility of mix up of camel swabs withthose of other domestic livestock. Those RT-PCR posi-tive animals were MERS-CoV seronegative. None ofthe swabs collected from sheep or goats in Tunisiawas rtRT-PCR positive.

In order to provide additional confirmation of thespecificity of the rtRT-PCR positive results for MERS-CoV RNA, the region of the RT-PCR amplified spikeRBD domain was sequenced. Sequence alignment ofpositive Egyptian samples from goats, cattle, and don-keys collected in 2015 or 2016 against the first humanMERS isolate (EMC/2012) which was used as the posi-tive control in RT-PCR revealed a point mutationA431 T in the S(RBD) gene (Figure S1). This sequenceheterogeneity reduces the possibility of PCR contami-nation as an explanation for RT-PCR positivity.

Alignment of the DPP4 sequences of various dom-estic mammalian species revealed that DPP4 residues

Table 1. RNA detection in nasal swabs and sera tested for MERS-CoV antibodies by microneutralization (VMN) and confirmed by90% plaque reduction neutralization (PRNT90) in samples obtained from domestic mammals in contact with camels in Egypt andTunisia.Species Number of nasal swabs MERS CoV positive Serum samples tested by VMN VMN positive Serum samples Tested by PRNT PRNT positive

EgyptSheep 191 2 195 1 0 –Goats 112 4 107 3 3 1Cattle 53 1 52 4 1 0Horse 6 0 6 0 – –Donkey 42 3 41 3 3 0Buffalo 5 0 5 0 – –TunisiaSheep 114 0 114 3 3 2Goats 204 0 204 5 3 0Cattle 15 0 15 0 – –Horse 2 0 2 1 0 –Donkey 2 0 2 0 – –Mule 5 0 5 0 – –SenegalSheep 63 1 63 35 35 35Goats 9 1 9 1 1 0

Table 2. Detection of MERS CoV RNA in nasal swabs in Egypt.Sample upE Orf1a Nseq S (RBD)

MERS CoV/donkey/Egypt/2213/2015 Pos Neg Neg PosMERS CoV/donkey/Egypt/3933/2016 Pos Neg Neg PosMERS CoV/donkey/Egypt/3976/2016 Pos Pos ND NDMERS CoV/cattle/Egypt/2257/2015 Pos Neg Neg PosMERS CoV/sheep/Egypt/3838/2016 Pos Pos Pos NDMERS CoV/sheep/Egypt/3853/2016 Pos Pos Pos NDMERS CoV/goat/Egypt/2284/2015 Pos Neg Neg PosMERS CoV/goat/Egypt/3850/2016 Pos Neg Neg PosMERS CoV/goat/Egypt/3893/2016 Pos Neg Neg PosMERS CoV/goat/Egypt/3975/2016 Pos Pos ND NDMERS CoV/sheep/Senegal/M46/2017 Pos Neg Pos NegMERS CoV/goat/Senegal/M178/2017 Pos Pos Neg Pos

Note: upE positive samples were confirmed by at least another assay target-ing the Orf1a, N, or S (RBD) genes. ND: Not done.

104 A. Kandeil et al.

interacting with the MERS CoV spike RBD were iden-tical in camel, sheep, and goat (Figure S2). EquineDPP4 had two amino acid mutations at positions 288(V288T) and 289 (P289A).

Discussion

MERS-CoV is a zoonotic virus and dromedary camelsare a source of human infection [10]. While there havebeen some surveillances of other domestic livestockspecies during the initial search for the natural hostof MERS-CoV in 2013-14, there is a paucity of a sys-tematic investigation of livestock in close proximityto infected dromedaries. We have previously reporteda seropositive sheep raised in contact with camels inEgypt [11]. However, surveillance in mammals in anarea where MERS-CoV infected camels in Jordanyielded negative results [7]. MERS-CoV infection wasnot detected in equids from the United Arab Emirateswhere camel and human infection were previouslyreported [9]. Although a study of horses in Saudi Ara-bia found no convincing evidence of MERS-CoV infec-tion in these horses, one of 243 equine sera had aPRNT90 titre of 1:40 [8].

In this paper, we provide the first convincing evi-dence of naturally acquired MERS-CoV infection insheep raised in prolonged close proximity with dro-medary camels in Senegal with a PRNT90 seropreva-lence of 55.6% with PRNT90 antibody titres rangingfrom 1:80 to ≥1:320. Evidence of PRNT90 antibodywas also found in 2 of 114 sheep sera collected fromTunisia and 1 of 112 goats in Egypt. Since not allVMN-positive sera from Egypt had sufficient serumto be tested in the confirmatory PRNT90 assay, wemay have under-estimated the true sero-prevalence inlivestock sampled in Egypt. The serological testing forMERS-CoV was carried out using a virus isolated inEgypt (camel/Egypt/163/14) (for the MN test) and pro-totype strain EMC (for the PRNT test). We have pre-viously shown that MERS-CoV from East, West, andNorth Africa are antigenically indistinguishable fromthose in the Arabian Peninsula and prototype strainEMC [12].

We have previously shown that antisera to closelyrelated betacoronaviruses such as bovine coronavirus,mouse hepatitis virus, or SARS coronavirus do notcross-react with MERS-CoV in neutralization tests[13]. Nevertheless, because BCoV is a betacoronavirusknown to be endemic in ruminants including sheep, wetested for VMN antibody to BCoV in selected MERS-CoV antibody positive sheep sera [14]. Our datasuggest that cross-reactive BCoV antibody does notexplain the observed antibody titres to MERS-CoV.The possibility of a hitherto undiscovered coronaviruscross-reacting with MERS-CoV cannot however beexcluded. Our findings may be evidence of repeatedcross-species transfer of MERS-CoV from dromedaries

to sheep rather than natural self-sustained trans-mission between sheep.

We detected MERS-CoV RNA by rtRT-PCR insheep, goats, and donkeys in farms in Egypt and in asheep and goat in nomadic herds in Senegal, all ofthese animals being in close contact with dromedaries.These animals were sero-negative, as expected, as sero-logical responses only appear 2–3 weeks after infectionby which time virus RNA will usually be undetectable.The rtRT-PCR positive animals were asymptomatic asis the case for camels infected with MERS-CoV. Theidentity of the host species of the swabs positive forvirus RNA was additionally confirmed by DNA bar-coding in order to assure that no mix up with camelswabs had occurred during sampling or sample hand-ling. Finding MERS-CoV RNA in upper respiratorytract swabs may simply represent exposure andenvironmental contamination from infected camelsor environments contaminated with camel excretionsrather than convincing evidence of active infection.In contrast, evidence of MERS-CoV neutralizing anti-body implies evidence of active infection by MERS-CoV or a closely related virus. Attempts to culturethe viruses detected in the study or to obtain full-gen-ome sequences were not successful hence limiting ourability to further verify the findings of this study. It isimportant to note that this evidence comes fromcountries where human infection with MERS-CoVwas never reported locally and that evidence ofMERS-CoV infection in non-camelid mammals wasnever reported in countries where human infectionsare common.

MERS-CoV was detected in species that expressDPP4 receptor essential for MERS-CoV attachmentand internalization into mammalian cells [6] and theDPP4 sequences of those species were similar to thatfound in camels, especially in regions where interactionwith the MERS-CooV spike protein RBD occurs. Twomutations were detected in the horse DPP4 sequences.

Interestingly, all sequences of MERS-CoV obtainedfrom the domestic livestock in contact with camels inEgypt possessed mutation A431T in the S gene(RBD) that was also detected in a sequence from a con-tact camel. Experimental inoculation of sheep andhorses with the first prototype isolate of MERS-CoV(HCoV-EMC/2012) showed no infection despite thatthe same virus was able to infect pigs and llamas[15,16]. This may be due to the absence of theA431T mutation in the virus used in those studies.This mutation may be a key to enabling MERS-CoVinfection in non-camelid domestic species and thisrequires further study. Future experimental infectionsof livestock need to be conducted also using MERS-CoV of African origin.

In summary, we provide evidence of MERS-CoV, ora very closely related virus, infection of domestic live-stock (other than camels) in close contact with camels,

Emerging Microbes & Infections 105

suggesting that spill over infection to other livestockmay occur. Our data does not prove MERS-CoV canbe sustained by transmission within these other live-stock species in the absence of camels as the primarysource of infection. However, if other domestic live-stock such as sheep may be infected by MERS-CoV,they may also be a risk factor for human infection.Our findings highlight the need for further field studiesof domestic livestock, especially those that are in closecontact with dromedary camels and also the need forexperimental studies infecting livestock such as sheepusing MERS-CoV isolates from Africa. Our findingsmay raise the need to segregate other livestock fromdromedary camels in farms and live animal markets.

Materials and methods

Samples, detection, and sequencing

In Egypt, 406 sera and 409 nasal swabs were collectedfrom 315 adult (>1 year) and 92 juvenile cattle,sheep, goats, donkeys, buffaloes, and horses in closecontact with camels from farms in three governorates:Sharkia (Nile Delta region), Beheira (Northern Egypt),and Luxor (Southern Egypt) monthly between August2015 and September 2017. In Tunisia, 342 sera and 346nasal swabs were obtained from 321 adult and 25juvenile cattle, sheep, goats, donkeys, mules, and horsesin contact with camels at a major livestock market inSouthern Tunisia during December and January of2016 and 2017. In Senegal, 72 sera and 72 nasalswabs were collected from 61 adult and 11 juvenilesheep and goats in contact with pastoral camels inthe Northwest of the country during August 2017.

Nasal swabs were screened by real-time reversetranscription PCR (rtRT-PCR) targeting upstream ofE gene of MERS-CoV [17]. Positive samples wereconfirmed using first the Open Reading Frame (ORF)1a. When ORF1a testing was negative, the N gene-based PCR assay was then performed as recommendedby the WHO [18]. Samples not confirmed by ORF1a orN were tested by an in-house RT-PCR for the spikeprotein receptor binding domain (RBD) (see below)and PCR products to confirm specificity of the RT-PCR assay.

A partial fragment in spike RBD was amplified usingpre-RBD_MERS_F (GAA TCT GGA GTT TAT TCAGTT TCG T) and pre-RBD_MERS_R (ACG GCCCGA AAC ACC ATA G) primers in the first roundusing one step RT-PCR kit (QIAGEN, Germany).The PCR products were then subjected to a secondPCR using RBD_MERS_F: (CGA AGC AAA ACCTTC TGG CT) and RBD_MERS_R: (ATA TTC CACGCA ATT GCC TA) using Phusion High FidelityPCR Master Mix Kit (Thermo Scientific, USA). Thephylogenetic tree was constructed using MEGA6

programme by applying the neighbor-joining methodwith Kimura’s two-parameter distance model and1000 bootstrap replicates [19]. Sequence alignmentwas performed using BioEdit 7.0 (ref. [20]). DPP4amino acid reference sequences of camels, sheep,goats, cattle, horses, and donkeys were downloadedfrom GenBank and aligned using BioEdit.

Serology tests

Microneutralization: The methods used have beendescribed before [21]. MERS-CoV (strains: EMC andcamel/Egypt/163/14) and bovine coronavirus (BCoV)(ATCC BRCV-OK-0514-2) were used. Vero cells(ATCC CCL-81) were used for MERS-CoV andHRT-18G cells (obtained from ATCC) for BCoV.Serum dilutions were mixed with equal volumes of200 tissue culture infective dose 50 of virus and incu-bated for one hour at 37°C. The virus-serum mixturewas then added in quadruplicate to cell monolayersin 96-well microtitre plates. After one hour of adsorp-tion, the virus-serum mixture was removed and 150 μlof fresh culture medium was added to each well and theplates incubated at 37°C in 5% CO2 in a humidifiedincubator. A virus back-titration was performed with-out immune serum to assess input virus dose. Cyto-pathic effect (CPE) was read at three days post-infection for MERS-CoV and four days post-infectionfor BCoV. The highest serum dilution that completelyprotected the cells from CPE in half of the wells wasdefined as the neutralizing antibody.

Plaque reduction neutralization (PRNT): The PRNTassays were performed on heat inactivated sera in 24-well tissue culture plates in duplicate for each serumdilution as previously described [21]. Briefly, two-foldserum dilutions were incubated with 40–60 plaque-forming units of MERS-CoV (strain EMC) virus for 1h at 37°C. Then the virus and serum mixture wereadded to a pre-formed Vero cell monolayer and incu-bated for 1 hr at 37°C in a 5% CO2 incubator. Then,the supernatant was removed and the cells were over-laid with 1% agarose (SeaKem LE Agarose, Lonza,Switzerland) in cell culture medium (Minimum Essen-tial Medium with 2% foetal bovine serum). After threedays incubation the plates were fixed and stained. End-point PRNT antibody titres were defined as the highestserum dilutions that resulted at ≥50% (PRNT50) and≥90% (PRNT90) inhibition of the number of plaques,respectively. Only the PRNT90 data is shown becausethis is the more stringent end point for virusneutralization.

RBD sequencing

A partial fragment in the spike protein RBD wasamplified with pre-RBD_mers_F (GAA TCT GGA

106 A. Kandeil et al.

GTT TAT TCA GTT TCG T) and pre-RBD_mers_R(ACG GCC CGA AAC ACC ATA G) primers usinga One-step RT-PCR kit (QIAGEN, Hilden Germany),in 25 µl reactions [5 µl of 5X reaction buffer, 1 µldNTPs, (10 mM) 1 µl enzyme mix, 1.5 µl (10 µM) for-ward primer, 1.5 µl (10 µM) reverse primer, 10 µlddH2O, and 5 µl of sample RNA]. The PCR cycler con-ditions were 50°C for 30 min, 95°C for 15 min, then 45cycles of 94°C for 15 s, 50°C for 30 s and 72°C for 90 s,and finally 72°C for 10 min. The PCR product was thensubjected to a second PCR using RBD_mers_F (CGAAGC AAA ACC TTC TGG CT) and RBD_mers_R(ATA TTC CAC GCA ATT GCC TA) primers andPhusion High Fidelity PCR Master Mix Kit, (ThermoScientific, Waltham, MA, USA). A 25 µl reactionincluded 12.5 µl of 2X phusion master mix, 1.5 µl(10 µM) forward primer, 1.5 µl (10 µM) reverse pri-mer, 6.5 µl H2O, and 3 µl of PCR product of the firstround. The PCR cycler conditions were 98°C for 30 s,then 40 cycles (98°C for 10 s, 48°C for 30 s, 72°C for60 s), then 72°C for 10 min. The final PCR productwas gel purified then, sequenced with the same primersat the Macrogen sequencing facility (Macrogen, SouthKorea).

DPP4 amino acid sequences of camels, sheep, goat,cattle, horse, and donkey (accession numbersAIG55259, AIG55264, AIG55261, NP776464,XP005601601, and XP014715582, respectively) weredownloaded from GenBank and aligned using BioEdit.

Host DNA barcoding

Cytochrome oxidase subunit 1 barcoding was used toconfirm species from which MERS CoV positiveswabs were obtained as previously described [22].The amplifed PCR products were sequenced andsequences were compared with available sequences inGenBank. Ethical approval was received from theSt. Jude Children’s Research Hospital IACUC.

Data availability

The nucleotide sequences obtained in this study areavailable from GenBank under accession numbersMF318497, MF318498, MF318499, MF318500,MF318501, MF318502, MF318503, and MF318504.

Acknowledgements

The funding agencies had no role in writing the manuscript.The authors wish to thank all personnel in Egypt and Tunisiawho facilitated and assisted with sample collection. AK, MG,MS, AN, AEK, AA, JJ, YM, OK, OB, SM, AM, RES, BE,RLWK, RAMP, and HS conducted the study. AK, NH, BE,MA, LA, AS, HS, PPM, RJW, MAA, and GK designed thestudy. AK, MAA, MP, and GK analysed the data and draftedthe manuscript.

Disclosure statement

No potential conflict of interest was reported by the authors.

Funding

This work was supported by the National Institute of Allergyand Infectious Diseases, National Institutes of Health,Department of Health and Human Services, under contractnumber HHSN272201400006C and by the United StatesAgency for International Development (USAID) in the fra-mework of OSRO/EGY/505/USA, through project jointlyimplemented by the Food and Agriculture Organization ,General Organization for Veterinary Services, and NationalResearch Center.

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